RNA binding proteins (RBPs) interact with functional RNA elements embedded within pre- and mature messenger RNA to form messenger ribonucleoprotein (mRNP) complexes. These interactions result in the faithful execution of RNA processing events such as pre-mRNA alternative splicing, RNA stability and translational control. Aberrant alterations in the interactions between the RBPs and their RNA elements ultimately lead to behavioral abnormalities and neurological developmental defects, which can often manifest as fatal diseases such as Spinal Muscular Atrophy and Amyotrophic Lateral Sclerosis or life-long debilitating behavioral abnormalities such as Prader-Willi/Angelman Syndromes, Schizophrenia and Autism Spectrum Disorder. These findings underscore the importance of investigating the roles of these RBPs in the brain. In particular, our project is aimed at systematically, using genome-wide biochemical and bioinformatic assays, identifying the functional RNA elements that are recognized by RBPs in mouse brain and human neurons. We will develop a novel resource of human pluripotent stem cells stably expressing tagged RBPs that can be differentiated into human neurons. This will enable the identification of RNA binding sites of 50 RBPs in human neural RNAs in a uniform and systematic manner using cutting-edge genomic approaches such as cross-linking and immunoprecipitation followed by high-throughput sequencing (CLIP- seq). To reveal the splicing, stability, and translational changes that are dependent on direct binding of these RBPs we will perform high-throughput sequencing of mRNAs (RNA-seq) and ribosome-protected fragments (RPFs). Finally, we will leverage our computational expertise to build predictive models using this genome-wide, multi-scale, mRNP code in the brain. PUBLIC HEALTH RELEVANCE: Over the last decade, defects in RNA binding proteins (RBPs) themselves or the interactions between RNA binding sites of RBPs have become recognized as increasingly responsible for neurological disease and mental disorders. This proposal seeks to establish the interactions among selected RBPs and their functional RNA elements in mammalian neurons. This will be a crucial first step in elucidating the RNA networks regulated by these RBPs in the brain.